Methods and compositions for liver specific delivery of therapeutic molecules using recombinant AAV vectors

ABSTRACT

Provided are methods for selectively expressing therapeutic molecules, such as secretory proteins, antisense molecules and ribozymes, in the liver. The methods find use in treating hepatic diseases or conditions. The methods also find use in treating any disease or condition in which systemic administration of the therapeutic substance, for example, a secretory protein, is desired. The methods involve administering to a mammalian patient having a need for liver expression of a therapeutic molecule an AAV vector containing a therapeutically effective amount of the therapeutic molecule. Also provided are novel vectors employable in these methods.

BACKGROUND

[0001] The therapeutic treatment of diseases and disorders by genetherapy involves the transfer and stable insertion of new geneticinformation into cells. Although a variety of physical and chemicalmethods have been developed for introducing exogenous DNA intoeukaryotic cells, viruses have generally been proven to be moreefficient for this purpose. Several DNA-containing viruses, such asparvoviruses, adenoviruses and herpesviruses, and RNA-containingviruses, such as retroviruses, have been used to construct eukaryoticcloning and expression vectors and explored as gene therapy vehicles.

[0002] Retrovirus and adenovirus based vectors are associated withcertain complications and disadvantages. For example, retroviruses areintimately associated with neoplastic events. See Donahue, Helper virusinduced T cell lymphoma in non-human primates after retroviral mediatedgene transfer, J Exp. Med. 176 (1992) 1125-1135. Adenovirus induces aCTL response. See Yang, MHC class 1-restricted cytotoxic T lymphocytesto viral antigens destroy hepatocytes in mice infected with El-deletedrecombinant adenoviruses, Immunity 1 (1994) 433-442. It also requires arelatively large (35 kb) viral genome, making its usefulness as avehicle to deliver large sequences limited.

[0003] Thus, an alternative vector which is neither pathogenic norimmunogenic would be advantageous. In contrast to adenoviruses, theparvovirus, adeno-associated virus (AAV), has a much smaller genome,most of which can be replaced by, foreign DNA. Parvoviruses are small,icohedral viruses approximately 25 nm in diameter containing a singlestrand DNA genome of approximately 5 kilobases (kb). They consist of twomajor classes: the dependoviruses, including AAV and its subtypes (AAV1,AAV2, AAV3, AAV4and AAV5), and the autonomous parvoviruses. The latterlytically infect permissive, proliferating cells in nonintegratingmanner without helper virus assistance. On the other hand, AAV is anon-pathogenic human parvovirus that requires co-infection with a helpervirus, usually adenovirus (or herpesvirus), for its optimal replication.See for example, Berns, Parvovirus replication, Microbiol. Rev. 54(1990) 316-329 and Berns and Bohenzky, Adeno-associated viruses: anupdate, Adv. Virus Res. 32 (1987) 243-306.

[0004] In the absence of a helper virus, the wild-type (wt) AAV has beenshown to integrate into the human chromosome 19 in a site-specificmanner. See Kotin and Berns, Organization of adeno-associated virus DNAin latently infected Detroit 6 cells, Virol. 170 (1989) 460-467; Kotin,Mapping and direct visualization of a region-specific viral DNAintegration site on chromosome 19q13-qter, Genomics 10 (1991) 831-834;Kotin, Site-specific integration by adeno-associated virus, Proc. Natl.Acad. Sci. 87 (1990) 2211-2215 and Samulski, Targeted integration ofadeno-associated virus (AAV) into human chromosome 19, EMBO J 10 (1991)3941-3950. Recombinant AAV vectors appear to lack this site-specificityof integration. See Ponnazhagan, Adeno-associated virus 2-mediatedtransduction of murine hematopoietic cells and long-term expression of ahuman globin gene in vivo, 6th Parvovirus Workshop, Montpellier, France.p29, (1995). Nevertheless, it has been suggested that the AAV-basedvector system may prove to be a safer alternative to the more commonlyused retrovirus- and adenovirus-based vectors. See, for example,Muzyczka, Use of adeno-associated virus as a general transduction vectorfor mammalian cells, Curr. Top. Microbiol. Immunol. 158 (1992) 97-129.Because approximately 90% of the human population is sero-positive forAAV (see, for example, Blacklow, A sero-epidemiologic study ofadeno-associated virus infection in infants and children, Am. J.Epidemiol. 94 (1971) 359-366), accidental infection by recombinant AAVis not likely to be problematic. Furthermore, relatively higherstability, higher titers, and higher transduction efficiency of AAV haveadded to the desirable features of AAV vectors. See Carter,Adeno-associated virus vectors, Curr. Opin. Biotechnol. 3 (1993) 533-538and Srivastava, Parvovirus-based vectors for human gene therapy, BloodCells 20 (1994) 531-538.

[0005] A number of studies have reported AAV-mediated successfultransduction and expression of therapeutic genes in vitro. For example,see Chatterjee, Dual target inhibition of HIV-1in vitro by means of anadeno-associated virus antisense vector, Science 258 (1992) 1485-1488;Walsh, Regulated high level expression of a human γ-globin geneintroduced into erythroid cells by an adeno-associated virus vector,Proc. Natl. Acad. Sci. 89 (1992) 7257-7261; Walsh, Phenotypic correctionof Fanconi anemia in human hematopoietic cells with a recombinantadeno-associated virus vector, J. Clin. Invest. 94 (1994) 1440-1448;Flotte, Expression of the cystic fibrosis transmembrane conductanceregulator from a novel adeno-associated virus promoter, J. Biol. Chem.268 (1993) 3781-3790; Ponnazhagan, Suppression of human α-globin geneexpression mediated by the recombinant adeno-associated virus 2-basedantisense vectors, J Exp. Med. 179 (1994) 733-738; Miller, Recombinantadeno-associated virus (rAAV)-mediated expression of human γ-globin genein human progenitor-derived erythroid cells, Proc. Natl. Acad. Sci. 91(1994) 10183-10187; Einerhand, Regulated high-level human beta-globingene expression in erythroid cells following recombinantadeno-associated virus-mediated gene transfer, Gene Ther. 2 (1995)336-343 Luo, Adeno-associated virus 2-mediated gene transfer andfunctional expression of the human granulocyte-macrophagecolony-stimulating factor, Exp. Hematol. 23 (1995) 1261-1267 and Zhou,Adeno-associated virus 2-mediated transduction and erythroidcell-specific expression of a human β-globin gene, Gene Therapy 3 (1996)223-229.

[0006] A few studies have examined the safety and efficacy of the AAVvectors in vivo (see Flotte, Stable in vivo expression of the cysticfibrosis transmembrane conductance regulator with an adeno-associatedvirus vector, Proc. Natl. Acad. Sci. 90 (1993) 10613-10617 and Kaplitt,Long-term gene expression and phenotypic correction usingadeno-associated virus vectors in the mammalian brain, Nature Genet. 8(1994) 148-153).

[0007] A disadvantage of AAV vectors in some clinical indications is thegeneralized nature of AAV infection. Previous studies have indicatedthat AAV possesses a wide host range that transcends the speciesbarrier. See for example Muzyczka, Use of adeno-associated virus as ageneral transduction vector for mammalian cells, Curr. Top. Microbiol.Immunol. 158 (1992) 97-129. The autonomous parvovirus, LuIII, appears topossess a similarly wide host range, since liver specific expression hasbeen obtained only via use of recombinants containing a liver-specificenhancer and a regulated promoter. See Maxwell, Autonomous parvovirustransduction of a gene under control of tissue-specific or induciblepromoters, Gene Therapy 3 (1996) 28036. Surprisingly, we have discoveredthat AAV exhibits organ tropism for the liver and is therefore uniquelyadapted for the treatment of diseases or conditions of the liver,diseases or conditions characterized by involving a protein made in theliver or diseases or conditions in which systemic administration of atherapeutic via the liver is desirable or advantageous.

INVENTION SUMMARY

[0008] In one aspect, the invention provides methods for selectivelyexpressing therapeutic molecules, such as secretory proteins, antisensemolecules and ribozymes, in the liver. The methods find use in treatinghepatic diseases or conditions. The methods also find use in treatingany disease or condition in which systemic administration of thetherapeutic substance, for example a secretory protein, is desired. Themethods also find use in treating or diseases or conditions involvingproteins that originate or are normally made in the liver.

[0009] The methods involve administering to a mammalian patient having aneed for liver expression of a therapeutic molecule a therapeuticallyeffective amount of an AAV vector containing a the therapeutic molecule.Therapeutic molecules useful in treating hepatic diseases or conditionswhich can be administered employing the methods described here include,for example, insulin and thymidine kinase,. Therapeutic moleculescomprising proteins originating in the liver or protein normally made inthe liver include, for example, the LDL receptor, Factor VIII, FactorIX, phenylalanine hydroxylase (PAH), ornithine transcarbamylase (OTC),and α1-antitrypsin. Therapeutic molecules comprising secretory proteinsin which systemic administration is advantageously attained via liverspecific delivery include, for example, cytokines, growth factors andthe colony stimulating factors, G-CSF and GM-CSF. Additional proteintherapeutic molecules contemplated for use in the methods andcompositions of the invention are described infra.

[0010] Also included are nucleic acid sequences that encode antisensemolecules that are useful in treating a hepatic disease. The antisensemolecule will be an RNA sequence that can prevent or limit theexpression of over-produced, defective, or otherwise undesirablemolecules by being sufficiently complementary in sequence to the targetsequence that it binds to the target sequence. For example, the targetsequence can be part of the mRNA that encodes a protein, and theantisense RNA would bind to the mRNA and prevent translation. The targetsequence can be part of a gene that is essential for transcription, andthe antisense RNA would bind to the gene segment and prevent or limittranscription. For example, Group C adenoviruses Ad2 and Ad5 have a 19kiloDalton glycoprotein (gp 19) encoded in the E3 region of the virusthat binds to class I MHC molecules in the endoplasmic reticulum ofcells and prevents terminal glycosylation and translation of themolecule to the cell surface. Prior to liver transplantation, the livercells may be infected with gp 19encoding AAV vectors or virions whichupon expression of the gp 19 inhibit the surface expression of class IMUC transplantation antigens. These donor cells may be transplanted withlow risk of graft rejection and may require a minimal immunosuppressiveregimen for the patient. It may also permit a donor-recipient state toexist with fewer complications.

[0011] Similar treatments may be used to treat chronic hepatitis Binfections or non-A non-B hepatitis. The vector can be engineered toinclude a structural hepatitis gene, polyadenylation signal or afragment thereof in reverse orientation such that the expression productbinds to hepatitis virus mRNA transcripts, preventing translation of thestructural protein and ultimately “inactivating” the virus. See, forexample, Wu, Specific inhibition of hepatitis B viral gene expression invitro by targeted antisense oligonucleotides, J. Biol. Chem. 267 (1992)12436-12439 and Offensperger, In vivo inhibition of duck hepatitis Bvirus replication and gene expression by phosphorothioate modifiedantisense oligodeoxynyucleotides, EMBO J 12 (1993) 1257-1262.

[0012] Also included are nucleic acid sequences that encode ribozymesthat are useful in treating various diseases and conditions. Ribozymesare RNA polynucleotides capable of catalyzing RNA cleavage at a specificsequence and hence useful for attacking particular mRNA molecules. Inchronic myelogenous leukemia for example, the “Philadelphia chromosomaltranslocation” causes expression of a bcr-abl fusion protein andabnormal function of the abl oncoprotein. Because the fusion mRNA occursonly in cells that have undergone the chromosome tanslocation andbecause the fusion transcript contains only two possible sequences atthe splice junction, a ribozyme specific for either of the two bcr-ablfusion mRNA splice junctions can inhibit expression of the oncoprotein.Exemplary ribozymes include ribozymes to hepatitis A, hepatitis B andhepatitis C. See Christoffersen and Marr, J. Med. Chem. 38 (1995)2023-2037 and Barpolome, J. Hepatol. 22 (1995) 57-64.

[0013] Currently preferred therapeutic molecules are the LDL receptor,Factor VIII, Factor IX, PAH, TPO (thrombopoietin) and EPO(erythropoietin). Also preferred are growth factors and cytokines. Atherapeutically effective amount of the therapeutic molecule forpurposes of this invention is at least about 10⁹ to about 10¹¹particles/body. The patient may be any mammal, although it iscontemplated that primate patients, and especially human patients, willbenefit most from the methods of treatment. Other patients may includemurine, canine, feline, bovine and equine species.

[0014] We contemplate that any AAV vector can be employed in the methodsof this invention. Leading and preferred examples of such vectors foruse in this invention are the AAV-2 basal vectors disclosed inSrivastava, PCT Patent Publication WO 93/09239. Most preferred are thevectors of the invention as disclosed herein. Such vectors comprise thetwo AAV ITRs (inverted terminal repeats) in which the authentic (i.e.,native) D-sequences of the ITRs are modified by the substitution ofnucleotides such that at least 5 authentic nucleotides and up to 18authentic nucleotides, preferably at least 10 authentic nucleotides upto 18 authentic nucleotides, most preferably 10 authentic (i.e., native)nucleotides, are retained and the remaining nucleotides of theD-sequence are deleted or replaced with non-native, i.e., exogenousnucleotides. One preferred sequence of 5 native nucleotides that areretained is 5′ CTCCA 3′. The authentic (i.e., native) D-sequences of theAAV ITRs are sequences of 20 consecutive nucleotides in each AAV ITR(i.e., there is one sequence at each end) which are not involved in HPformation. The exogenous or non-native replacement nucleotide may be anynucleotide other than the nucleotide found in the native D-sequence inthe same position. For example, appropriate replacement nucleotides fornative D-sequence nucleotide C are A, T and G and appropriatereplacement nucleotides for native D-sequence nucleotide A are T, G andC. The construction of four such vectors is exemplified in Example 4, towit, preferred vectors pD-5, pD-15 and pD-20, and most preferred vectorpD-10, using the vector pXS-22 as starting material.

[0015] Other employable exemplary vectors are pWP-19, pWN-1 both ofwhich are disclosed in Nahreini, Gene 124 (1993) 257-262. Anotherexample of such an AAV vector is psub201. See Samulski, J. Virol. 61(1987) 3096. Another example is the Double-D ITR vector. How to make theDouble-D ITR vector is disclosed in U.S. Pat. No. 5,478,745. Still othervectors are those disclosed in Carter, U.S. Pat. No. 4,797,368 andMuzyczka, U.S. Pat. No. 5,139,941, Chartejee, U.S. Pat. No. 5,474,935,and Kotin, PCT Patent Publication WO 94/28157. Yet a further example ofan AAV vector employable in the methods of this invention isSSV9AFABTKneo, which contains the AFP enhancer and albumin promoter anddirects expression predominantly in the liver. Its structure and how tomake it are disclosed in Su, Selective killing of AFP-positivehepatocellular carcinoma cells by adeno-associated virus transfer of theherpes simplex virus thymidine kinase gene, Human Gene Therapy 7 (1996)463-470. The disclosures of these scientific articles, U.S. Patents andpatent publications are herein incorporated by reference.

[0016] Although not an absolute requirement for the practice of theinvention, in a further embodiment, the AAV vectors of the invention maycontain a liver specific promoter to maximize the potential for liverspecific expression of the exogenous DNA sequence contained in thevectors. The promoter is operably linked to the nucleic acid encodingthe therapeutic molecule upstream from the latter and between the AAVvector sequences (for example between the inverted terminal repeats inpsub201 or downstream of the Double D ITR sequence) Preferred liverspecific promoters include the hepatitis B X-gene promoter and thehepatitis B core protein promoter. These liver specific promoters arepreferably employed with their respective enhancers. The enhancerelement can be linked at either the 5′ or the 3′ end of the nucleic acidencoding the therapeutic molecule. The hepatitis B X gene promoter andits enhancer can be obtained from the viral genome as a 332 base pairEcoRV-NcoI DNA fragment employing the methods described in Twu, J Virol.61 (1987) 3448-3453. The hepatitis B core protein promoter can beobtained from the viral genome as a 584 base pair BamHI-BgIII DNAfragment employing the methods described in Gerlach, Virol 189 (1992)59-66. It may be necessary to remove the negative regulatory sequence inthe BamHI-BgIII fragment prior to inserting it. Other liver specificpromoters include the AFP (alpha fetal protein) gene promoter and thealbumin gene promoter, as disclosed in EP Patent Publication 0 415 731,the α-1 antitrypsin gene promoter, as disclosed in Rettenger, Proc.Natl. Acad. Sci. 91 (1994) 1460-1464, the fibrinogen gene promoter, theAPO-A1 (Apolipoprotein A1) gene promoter, and the promoter genes forliver transference enzymes such as, for example, SGOT, SGPT andγ-glutamyle transferase. See also PCT Patent Publications WO 90/07936and WO 91/02805.

[0017] We also contemplate that any hepatic disease or any defect inhepatic function, whether inherited or acquired, is susceptible totreatment with the methods of the invention. Exemplary hepatic diseasesor defects in hepatic function include hepatocellular carcinoma,jaundice, infectious hepatitis, alcohol liver damage, including alcoholinduced cirrhosis, and non-alcohol induced liver cirrhosis.

[0018] We also contemplate that any inherited or acquired disease ordefect, the treatment of which requires administration of a therapeuticmolecule that is normally made in the liver, is susceptible to treatmentwith the methods of the invention. Exemplary inherited diseases includefamilial hypercholesterolemia, which is caused by an LDL receptordeficiency, phenylketonuria, which is caused by a phenylalaninehydroxylase deficiency, urea cycle disorders, organic acid disorders,Wilson's disease, tyrosinemia, α₁-antitrypsin deficiency andhyperammonemia which is caused by an inherited deficiency of ornithinetranscarbamylase d function. Exemplary acquired diseases includenon-familial hypercholesterolemia and other hyperlipoproteinemias.

[0019] We also contemplate that the methods described here find use intreating any disease or condition in which the therapeutic substance,for example a secretory protein, is advantageously expressed in theliver in order to, for example, obtain systemic administration via entryinto the circulatory system through the hepatic system. Genes encodingany of the cytokines and immunomodulatory proteins described here can beexpressed in an AAV vector to achieve liver specific in vivo expression.Forms of these cytokines other than the forms mentioned here that areknown to the skilled artisan can be used. For instance, nucleic acidsequences encoding native IL-2 (interleukin 2) and γ-interferon can beobtained as described in U.S. Pat. Nos. 4,738,927 and 5,326,859respectively, while useful mutants of these proteins can be obtained asdescribed in U.S. Pat. No. 4,853,332. As an additional example, nucleicacid sequences encoding the short and long forms of M-CSF (macrophagecolony stimulating factor) can be obtained as described in U.S. Pat.Nos. 4,847,201 and 4,879,227 respectively. AAV vectors expressingcytokine or immunomodulatory genes can be produced as described here.

[0020] AAV vectors producing a variety of known polypeptide hormones andgrowth factors can be used in the methods of the invention to producetherapeutic expression of these proteins. Some such hormones, growthfactors and other proteins are described in EP patent 0 437 478 B1forinstance. Nucleic acid sequences encoding a variety of hormones can beemployed, including for example, human growth hormone, insulin,calcitonin, prolactin, follicle stimulating hormone, luteinizinghormone, human chorionic gonadotropin, thyroid stimulating hormone. AAVvectors expressing polypeptide hormones and growth factors can beprepared by methods known to those of skill in the art. As an additionalexample, nucleic acid sequences encoding different forms of humaninsulin can be isolated as described in EP patent publication 026598 or070632 and incorporated into AAV vectors as described here.

[0021] Any of the polypeptide growth factors can also be administeredtherapeutically by liver specific expression in vivo with an AAV vector.For instance, different forms of IGF-1 and IGF-2 growth factorpolypeptides are well known in the art and can be incorporated into AAVvectors for liver specific expression. See EP patent 0 123 228 B1. Liverspecific expression of different forms of fibroblast growth factor canalso be effected by the methods of the invention. See U.S. Pat. Nos.5,464,774; 5,155,214 and 4,994,559.

[0022] There are a number of proteins useful for treating hereditarydisorders that can be expressed by the methods of the invention. Manygenetic diseases caused by inheritance of defective genes result in thefailure to produce normal gene products, for example, severe combinedimmunodeficiency (SCID), hemophilia A, hemophilia B, adenine deaminasedeficiency, Gaucher's syndrome, hereditary lactose intolerance andinherited emphysema. Also contemplated are diseases that are caused bythe inability of the gene to produce adequate levels of the appropriatehormone, such as diabetes and hypopituitarism.

[0023] Liver specific expression of Factor VIII or Factor IX, useful forthe treatment of blood clotting disorders such a hemophilia, isobtainable using the methods of the invention. PCT Patent Publication WO96/21014 describes Factor VIII and HGH (human growth hormone) constructsfor retroviral expression which could readily adapted by the skilledartisan for AAV expression. The Factor VIII minigene (see EP PatentPublication 232 112 and PCT Patent Publication WO 91/07490) couldadvantageously be employed for AAV expression. Also contemplated is theexpression of lactase for the treatment of hereditary lactoseintolerance, ADA for the treatment of ADA deficiency and α-1 antitrypsinfor the treatment of α-1 antitrypsin deficiency. See Ledley, JPediatrics 110: (1987) 157-174; Verma, Scientific American (Nov. 1987)pp. 68-84 and PCT Patent Publication WO95/27512.

[0024] There are a variety of other proteins of therapeutic interestthat can be expressed in a liver specific manner using the methods ofthe invention. For instance sustained expression of tissue factorinhibitory protein (TFPI) is useful for the treatment of conditionsincluding sepsis and DIC and in preventing reperfusion injury. See PCTPatent Publications WO 93/24143, WO 93/25230 and WO 96/06637. Nucleicacid sequences encoding various forms of TFPI can be obtained, forexample, as described in U.S. Pat. Nos. 4,966,852; 5,106,833 and5,466,783, and can be incorporated into AAV vectors as described here.

[0025] Other proteins of therapeutic interest such as erythropoietin(EPO) and leptin can be expressed in the liver by AAV vectors accordingto the methods of the invention. EPO is useful in gene therapy treatmentof a variety of disorders including anemia. See PCT Patent PublicationWO 95/13376. Gene therapy delivery of the leptin gene and its use in thetreatment of obesity is described in PCT Patent Publication WO 96/05309.AAV vectors expressing EPO or leptin can readily be produced and liverspecific expression attained employing the described methods. Otherexemplary proteins and polypeptides include the cytokines such asinterleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14and IL-15, α-interferon, β-interferon,the γ-interferons, GM-CSF, the tumor necrosis factors (TNFs), CD3,ICAM-1, LFA-1, LFA-3, the chemokines including RANTES 1α, MIP-1α, MIP-1γ(see Cocchi, Science 720 (1996) 1811-1815) or analogs of such proteins.Because soluble forms of receptors can often behave as antagonists, ascan mutated forms of the factors themselves, the nucleic acid sequencesof therapeutic interest may also be agonists, antagonists or ligands forthese proteins and polypeptides.

[0026] Even more proteins and polypeptides of therapeutic interest thatcan be expressed in liver specific fashion employing the AAV vectors andmethods of the invention include Protein S and Gas6, thrombin,Coagulation Factor Xa, CSF-1 or M-CSF, IGF-1, IGF-2, acidic FGF, basicFGF, keratinocyte growth factor (KGF), TGF, platelet derived growthfactor (PDGF), epidermal growth factor (EGF), hepatocyte growth factor(HGF) and HGF activators, PSA, nerve cell growth factor (NCGF), glialcell derived nerve growth factor (GDNF), VEGF, Argvasopressin, thyroidhormones, azoxymethane, triiodothyronine, LIF, ampbiregulin, solublethrombomodulin, stem cell factor, osteogenic protein 1, the bonemorphogenic proteins, MGF, MGSA, heregulins and melanotropin. Growthfactors can also be used in combination with mixtures consisting of oneor several of, for example, DGF, IGF, PDGF, FGF or KGF. The full lengthgrowth factor can be employed or forms of the growth factor, such asactive fragments, truncated forms and analogues can be employed. By“active fragment” we mean a polypeptide containing less than afull-length sequence that retains sufficient biological activity to beused in the methods of the invention. By “analogue” we mean truncatedforms, splice variants, variants with amino acid substitutions,deletions or additions, alleles and derivatives of the mature protein orpolypeptide which possess one or more of the native bioactivities of thefull length protein or polypeptide. Thus, polypeptides that areidentical or contain at least 60%, preferably, 70% , more preferably 80%and most preferably 90% amino acid sequence homology to the amino acidsequence of the mature protein wherever derived, from human or non-humansources are included within this definition. For example, a preferredtruncated form of KGF is described in PCT Patent Publication WO95/10434. See also PCT Patent Publication WO 90/08771 and U.S. Pat. No.5,096,825 relating to human EGF.

[0027] The growth factor polypeptides, fragments and analogues can beproduced by isolation from naturally occurring sources, polypeptidechain synthesis by peptide synthesis methods and production orrecombinant proteins. These methods are well known to those of skill inthe art. For example, production of recombinant PDGF is described inU.S. Pat. Nos. 5,045,633 and 4,769,328 and production of recombinant FGFand analogues is described in U.S. Pat. Nos., 5,229,501; 5,331,095 and5,143,829.

[0028] A variety of other disorders can be treated by the methods of theinvention. For example, production of apolipoprotein E or apolipoproteinA, useful in treating hyperlipidemia, can be attained via administrationof the liver specific AAV vectors of the invention. See Breslow,Biotechnology 12 (1994) 365. Sustained production of angiotensinreceptor inhibitor (see Goodfriend, N. Engl. J. Med. 334 (1996) 1469) orof angiostatin useful in the treatment of tumors (see O'Reilly, NatureMed. 2 (1996) 689) can be attained.

[0029] Nucleic acid sequences that encode the above-described proteinsand polypeptides are obtainable from a variety of sources. For example,plasmids containing sequences the encode altered cellular products maybe obtained form a depository such as the American Type CultureCollection (ATCC, Rockville, Md.) or from commercial sources such asAdvanced Biotechnologies (Columbia, Md.) and British Bio-TechnologyLimited (Cowley, Oxford, Great Britain). Exemplary plasmids include ATCCNos. 41000 and 41049 containing muteins of ras. Other nucleic acidsequences that encode the above-described proteins and polypeptides, aswell as other nucleic acid molecules such as antisense sequences andribozymes that are advantageously used in the invention may be readilyobtained from such public sources. Exemplary are BBG12 containing thefull length GM-CSF coding sequence, BBG6 containing the γ-interferoncoding sequence, ATCC No. 39656 containing sequences encoding TNF, ATCCNo. 20663 containing sequences encoding α-interferon, ATCC Nos. 31902and 39517 containing sequences encoding β-interferon, ATCC No. 67024containing the interleukin-1b coding sequence, ATCC Nos. 39405, 39452,39516, 39626 and 39673 containing sequences encoding interleukin-2, ATCCNo. 57592 containing sequences encoding interleukin -4, ATCC Nos. 59394and 59395 containing sequences encoding interleukin-5 and ATCC 67153containing sequences encoding interleukin-6. Molecularly cloned genomesencoding the hepatitis B virus are obtainable from the ATCC. ATCC No.45020 contains the total genomic DNA of hepatitis B(with correctableerrors), extracted from purified Dane particles, in the BamHI site ofpBR322. See Blum TIG 5 (1989) 154-158 and Moriarty, Proc. Natl. Acad.Sci. 78 (1981) 2606-2610. Alternatively, cDNA sequences for use with theinvention are obtainable from cells that express or contain thesequences. Briefly, within one embodiment, mRNA from a cell thatexpresses the gene of interest is reverse transcribed with reversetranscriptase using oligo dT or random primers. The single stranded cDNAmay then be amplified by PCR (see U.S. Pat. Nos. 4,683,202; 4,683,195and 4,800,159, PCR Technology: Principles and Applications for DNAAmplification, Erlich (ed.), Stockton Press, 1989) using oligonucleotideprimers complementary to sequences on either side of desired sequences.In particular, a double stranded DNA is denatured by heating in thepresence of heat stable Taq polymerase, sequence specific DNA primers,ATP, CTP, GTP and TTP. Soluble-stranded DNA is produced when synthesisis complete. This cycle may be repeated many times resulting in afactorial amplification of the desired DNA. Nucleic acid sequences mayalso be synthesized de novo, for example on an Applied Biosystems Inc.DNA synthesizer.

[0030] In another embodiment, AAV hybrid (i.e., chimeric) vectors areprovided containing the DNA sequence, or functional fragment thereof,encoding hepatitis B surface antigen and the DNA sequence encoding theAAV capsid protein. An oligonucleotide sequence that corresponds to thisHBV surface antigen peptide is blunt-ended and ligated at the 5′ end ofthe AAV VP-1 gene. Specifically, the 27 amino acid sequence of HBVsurface antigen corresponding to amino acids 20-47 of the preS1 region(see, Ishikawa, Proc. Natl. Acad. Sci. 92 (1995) 6259-6263; Klingmuller,J Virol. 67 (1993) 7414-7422 and Neurath, Cell 46 (1986) 429-436 andVirol 176 (1990) 448-457) are fused in-frame to the AAV viral capsidVP-1 gene (see Srivastava, J Virol. 45 (1983) 555-564). Nucleic acidsencoding therapeutic molecules cloned within recombinant AAV vectors maybe packaged into recombinant AAV virions using this AAV-HBV chimerichelper vector. The AAV-2 capsid gene has been cloned and is available.See Samulski, J Virol. 63 (1989) 3822-3828. But capsid genes from anyAAV, specifically from AAV-1, AAV-3 or AAV-4, can be employed. For ageneral review of the molecular biology, structure and gene products ofHBV see Yoffe, Progress and perspectives in human hepatitis B virusresearch. Prog. Med. Virol. 40 pp. 107-140 (Melnick, J. L. ed.) 1993.

[0031] To establish integration of the vector into the chromosome of ahost cell, host cells are transfected with the vector or infected withmature virions containing the vector. Methods of transfection arewell-known in the art and include, for example, naked DNA transfection,microinjection and cell fusion. Virions can be produced by coinfectionwith helper virus such as adenovirus, herpes virus or vaccinia virus.Following coinfection with the vector and a helper virus, the host cellsare isolated and the helper virus is inactivated. The resulting helperfree stocks of virions are used to infect host cells. Alternatively,virions are produced by cotransfecting helper virus-infected cells withthe vector and a helper plasmid. The plasmid will contain the parvovirusrep gene and non-AAV ITRs, for example adenovirus ITRs. Followingcotransfection, mature virions are isolated using standard methods andany contaminating adenovirus inactivated using methods known to skilledartisans. The resulting mature virions can be used to infect host cellsin the absence of helper virus.

[0032] Methods of making recombinant AAV vectors and packaging celllines, purification methods, rescue methods and methods of generatinghigh-titer vector stocks are known in the art. See for example,Samulski, A recombinant plasmid from which an infectiousadeno-associated virus genome can be excised in vitro and its use tostudy viral replication, J. Virol 61 (1987) 3096-3101 and helper-freestocks of recombinant adeno-associated viruses: Normal integration doesnot require viral gene expression, J Virol. 63 (1989) 3822-3828,McLaughlin, Adeno-associated virus general transduction vectors:Analysis of proviral structures, J Virol. 62 (1988) 1963-1973, Flotte.An improved system for packaging recombinant adeno-associated virusvectors capable of in vivo transduction, Gene Therapy 2 (1995) 29-37,Holscher, Cell lines inducibly expressing the adeno-associated virus(AAV) rep gene: Requirements for productive replication of rep-negativeAAV mutants, J Virol. 68 (1994) 7169-7177 and High-level expression ofadeno-associated virus (AAV) Rep78 protein is sufficient forinfectious-particle formation by a rep-negative AAV mutant, J. Virol. 69(1995) 6880-6885, Yang, Characterization of cell lines that induciblyexpress the adeno-associated virus Rep proteins, J Virol. 68 (1994)4847-4856, Ponnazhagan, Alternative strategies for generatingrecombinant AAV vectors, VIth parvovirus Workshop, Montpellier, France,p. 71(1995), Luhovy, Stable transduction of recombinant adeno-associatedvirus into hematopoietic stem cells from normal and sickle cellpatients, Bio. Blood Marrow Transpl. 2 (1996) 24-30, Tamayose, A newstrategy for large-scale preparation of high-titer recombinantadeno-associated virus vectors by using sulfonated cellulose columnchromatography, Hum. Gene Therap. 7 (1996) 507-513, Maxwell, Improvedmethod for production of recombinant AAV and determination of infectioustiter, VIth Parvovirus Workshop, Montpellier, France, p72 (1995),Chiorini, High-efficiency transfer of the T cell co-stimulatory moleculeB7-2 to lymphoid cells using high-titer recombinant adeno-associatedvirus vectors, Hum. Gene Therap. 6 (1995) 1531-1541 and Colosi, AAVvectors can be efficiently produced without helper virus, Blood 10(1995) 627a.

[0033] The vector or virions can be incorporated into pharmaceuticalcompositions for administration to mammalian patients, particularlyhumans. The vector or virions can be formulated in nontoxic, inert,pharmaceutically acceptable aqueous carriers, preferably at a pH rangingfrom 3 to 8, more preferably ranging from 6 to 8. Such sterilecompositions will comprise the vector or virion containing the nucleicacid encoding the therapeutic molecule dissolved in an aqueous bufferhaving an acceptable pH upon reconstitution. Such formulations comprisea therapeutically effective amount of a AAV vector or virion inadmixture with a pharmaceutically acceptable carrier and/or excipient,for example saline, phosphate buffered saline, phosphate and aminoacids, polymers, polyols, sugar, buffers, preservatives and otherproteins. Exemplary amino acids, polymers and sugars and the like areoctylphenoxy polyethoxy ethanol compounds, polyethylene glycolmonostearate compounds, polyoxyethylene sorbitan fatty acid esters,sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran,sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine orhuman serum albumin, citrate, acetate, Ringer's and Hank's solutions,cysteine, arginine, carmitine, alanine, glycine, lysine, valine,leucine, polyvinylpyrrolidone, polyethylene and glycol. Preferably, thisformulation is stable for at least six months at 4° C.

[0034] The virions can be systemically administered by intravenousinjection. The dosage regimen will be determined by the attendingphysician or veterinarian considering various factors known to modifythe action of drugs such as, for example, the physical condition of thepatient, the severity of the condition, body weight, sex, diet, time ofadministration and other clinical factors. Generally, the regimen shouldbe in the range of about 10⁹ to about 10¹¹ particles per body. Apreferred dose is about 10¹⁰ particles per body. The number of dosesadministered may vary, depending on the above mentioned factors.

[0035] The AAV vector or virions can also be administered ex vivoemploying art recognized methods, for example, by electroporationfollowing the procedures of Chakrabarti, J. Biol. Chem. 264 (1989)15494-15500 or by protoplast delivery following the procedures ofKaneda, Science 243 (1989) 375-78 and Ferguson, J. Biol. Chem 261 (1986)14760-14763. Alternatively, hepatocyte precursor cells can be transducedwith a vector of the invention, grown in tissue culture vessels, removedand introduced into the patient surgically by grafting ortransplantation. The precursor cells can be attached to supports such asmicrocarrier beads that are injected into the peritoneal space of thepatient or directly into the liver, into the portal venous system orinto the spleen. The patient's liver cells may be obtained through liverbiopsy, partial hepatectomy or from specimens harvested for orthotopicliver transplantation, purified and grown in culture. AAV vectors may beintroduced into the liver cells by exposure to the virus and the livercells reintroduced into the patient by grafting or by placing the cellsin the abdominal cavity in contact with the unremoved portion of thepatient's liver. Such methods are known in the art. See, for example,Chang, Gene Therapy: Applications to the Treatment of Gastrointestinaland Liver Diseases, Gastroenterology 106 (1994) 1076-1084. For ex vivoadministration, the dosage regimen should be in the range of 1 to 100m.o.i., preferably in the range of 5 to 20 m.o.i. The dosage regimenwill be determined by the attending physician considering variousfactors known to modify the action of drugs such as for example,physical condition, body weight, sex, diet, severity of the condition,time of administration and other clinical factors. The number of dosesadministered may vary, depending on the above mentioned factors.

[0036] The liver specific delivery methods of the invention may beemployed with or without pretreatment of the liver. Pretreatmentincludes benign hyperplasia, which can be induced by treatment with HGFand/or transforming growth factors. See Lui, Hepatology 19 (1994) 1521.Different forms of HGF useful in inducing liver cell proliferation areknown in the art and can be employed. See for example EP patentpublication EP 0 461 560. HGF can also be produced and administered toinduce liver cell proliferation in vivo as described in Joplin, J. Clin.Invest. 90 (1992) 1284. Liver cells can also be stimulated byadministration of agents that mediate or potentiate the activation ofendogenous HGF. HGF is produced as a single chain protein that isinactive as a growth factor. Single chain HGF is subsequently cleavedinto a two-chain form that is biologically active. Enzymes that areshown to convert single-chain HGF to its bioactive form are useful forinducing liver cell proliferation. Therefore, these enzymes can beadministered either alone or in combination with exogenous HGF toenhance liver proliferation. Exemplary enzymes include coaglation factorXI1a, HGF activator, HGF converting enzyme, urokinase and tissueplasminogen activator. For example, HGF and urokinase can beco-formulated and administered by IV injection or mixed immediatedlyprior to injection. If co-formulated, storage at low pH wouldadvantageously minimize the activity of urokinase. See PCT PatentPublication WO 96/21014 entitled Production and Administration of HighTiter Recombinant Retroviruses.

[0037] In another embodiment of the invention the AAV vector isco-administered with a cholesterol lowering drug to a primate patientsuffering from hypercholesterolemia. A preferred cholesterol loweringdrug is M-CSF. See U.S. Pat. Nos. 5,021,239 and 5,019,381. Otherpreferred cholesterol lowering drugs include niacin, gemfibrozil,lovastatin and mevacor.

DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1: Illustration of the Southern blot analysis of thePCR-amplified DNA fragments of the vCMVp-lacZ vector administered tomice as described in Example 1, in various murine tissues.

[0039]FIG. 2: Illustration of the Southern blot analysis of thePCR-amplified DNA fragments of the vHS2-βp-^(A)γ-globin vectoradministered to mice as described in Example 2, in various murinetissues.

[0040]FIG. 3: Illustration of autoradiogram of semi-quantitative PCRamplification results, as detailed in Example 2 .

[0041]FIG. 4: Schematic structures of pSub201 and pD-10 recombinant AAVvectors, as detailed in Example 5. The D-sequence is shown as a shadedbox in plasmid pSub201. In plasmid pD-10, the distal 10 nucleotides inthe D-sequence have been replaced by a substitute (s)-sequence.

DETAILED DESCRIPTION

[0042] In murine mammalian patients the fate of AAV vectors was followedafter direct intravenous injection and it was surprisingly found thatthe AAV vectors possess organ-tropism for liver. Our AAV vectorscontained the lacZ reporter gene or the human globin gene. In miceadministered the lacZ reporter gene containing AAV vectors, expressionoccured in hepatocytes but a cytotoxic T lymphocyte response againstβGal was not detected. The recombinant AAV vectors, when directlyinjected intravenously in mice, accumulated predominantly in livercells.

[0043] The AAV recombinant virus stocks containing the CMV promoter(CMV_(p)) driven lacZ gene (vCMVp-lacZ) cloned in between AAV invertedterminal repeats (ITR) and the AAV recombinant virus stocks containingthe genomic copy of the normal human ^(A)γ-globin gene driven by thehuman β-globin promoter (βp) plus an upstream Hypersensitive site 2enhancer element cloned in between AAV ITR were generated from theirrespective recombinant plasmids by the methods described in Samulski,Helper-free Stocks of Recombinant Adeno-associated Viruses: NormalIntegration Does Not Require Viral Gene Expression, J Virol. 63 (1989)3822-3828; Nahreini,Versatile Adeno-associated Virus 2-based Vectors forConstructing Recombinant Virions, Gene 124 (1993) 257-262; Zhou,Adeno-associated Virus 2-mediated High Efficiency Gene Transfer intoImmature and Mature Subsets of Hematopoietic Progenitor Cells in HumanUmbilical Cord Blood, J Exp. Med. 179 (1994) 1867-1875; Ponnazhagan,Lack of Site-specific Integration of the Recombinant Adeno-associatedVirus Genomes in Human Cells, 5th Parvovirus Workshop, Crystal River,Fla., USA p.P1-29 (1993); Ponnazhagan, Adeno-associated Virus 2-mediatedTransduction of Murine Hematopoietic Cells and Long-term Expression of aHuman Globin Gene in Vivo, 6th Parvovirus Workshop, Montpellier, France.p29 (1995); and Ponnazhagan, Differential Expression in Human Cells fromthe P6 Promoter of Human Parvovirus B19 Following Plasmid Transfectionand Recombinant Adeno-associated Virus 2 (AAV2) Infection: HumanMegakaryocytic Leukaemia Cells Are Non-Permissive for AAV Infection, J.Gen. Virol. 77 (1996) 1111-1122. The viral stocks were purified on CsCldensity gradients following the protocol described in Wang, ParvovirusB19 Promoter at Map Unit 6 Confers Replication Competence and ErythroidSpecificity to Adeno-associated Virus 2 in Primary Human HematopoieticProgenitor Cells, Proc. Natl. Acad. Sci. 92 (1995) 12416-12420. Titerswere determined on quantitative DNA slot blots as described inSrivastava, Parvovirus B19-induced. Perturbation of HumanMegakaryocytopoiesis In Vitro, Blood 76 (1990) 1997-2004; Srivastava,Construction of a Recombinant Human Parvovirus B19: Adeno-associatedVirus 2 (AAV) DNA Inverted Terminal Repeats Are Functional in an AAV-B19Hybrid Virus, Proc. Natl. Acad. Sci. 86 (1989) 8078-8082.; Nahreini andSrivastava, Rescue of the Adeno-associated Virus 2 Genome Correlateswith Alterations in DNA-modifying Enzymes in Human Cells, Intervirol. 33(1992) 109-115.; Zhou, Adeno-associated Virus 2-mediated Gene Transferin Murine Hematopoietic Progenitor Cells, Exp. Hematol. 21 (1993)928-933; Zhou, Adeno-associated Virus 2-mediated High Efficiency GeneTransfer into Immature and Mature Subsets of Hematopoietic ProgenitorCells in Human Umbilical Cord Blood, J Exp. Med. 179 (1994) 1867-1875and Zhou, Adeno-associated Virus 2-mediated Transduction and ErythroidCell Specific Expression of a Human β-globin Gene, Gene Therapy 3 (1996)223-229.

[0044] These highly purified recombinant AAV vectors were administeredto C57B1/6 mice by direct intravenous injection into the tail vein.

EXAMPLE 1

[0045] Highly purified recombinant AAV vectors containing thecytomegalovirus (CMV) promoter-driven lacZ gene (vCMVp-lacZ) weredirectely injected into C57B1/6 mice. Approximately 1×10¹⁰ viralparticles of vCMVp-lacZ were injected intravenously into the tail-veinof 12 animals in four groups of three animals each. These animals weresacrificed at various times post-injection (p.i.), and equivalentamounts of tissues from various organs were examined for the presence ofthe recombinant AAV viral genome by polymerase-chain-reaction (PCR)amplification using a lacZ-specific primer-pair followed by Southernblot analysis.

[0046] Approximately 1×10¹⁰ particles of the vCMVp-lacZ r-virus wereinjected in 0.2 ml Iscove's-modified Dulbecco's medium into thetail-vein of 8-week old C57B1/6 mice. Three animals per group weresacrificed at 1 hour, 24 hours, 72 hours, and 1 week p.i. Individualtissues and organs were obtained, rinsed extensively withphosphate-buffered-saline, and equivalent amounts were used in a35-cycle PCR-amplification reaction using the lacZ-specific primer-pair(5′-GATGAGCGTGGTGGTTATG, 5′-TACAGCGCGTCGTGATTAG). Plasmids pCMVp-lacZ(Ponnazhagan et al., 1996) and pUC19 (Sambrook, Fritsch, and Maniatis,Molecular Cloning: A Laboratory Manual, CSHL Press, Cold Spring Harbor,N.Y., 1989) were used as positive and negative controls, respectively.The PCR products were electrophoresed on 1% agarose gels and analyzed onSouthern blots (Southern, Detection of specific sequences among DNAfragments separated by gel electrophoresis. J. Mol. Biol. 98 (1975)503-517) using a lacZ-specific ³²P-labeled DNA probe.

[0047] The results of the Southern blot analysis are shown in FIG. 1.The recombinant AAV genomes were detected predominantly in the livertissues up to 1-week p.i. in each group of animals. The arrows indicatethe 588-bp lacZ-specific DNA fragment.

EXAMPLE 2

[0048] The results in Example 1 were corroborated by injectingrecombinant vHS2-βp-^(A)γ-globin virions under conditions identical tothose in Example 1 and examining tissues from various organs seven weeksp.i. using the same techniques, but employing a β-globinpromoter-^(A)γ-globin gene-specific primer-pair.

[0049] Highly purified recombinant AAV vectors containing the humanβ-globin promoter-driven human ^(A)γ-globin gene containing the DNasehypersensitive-site 2 (HS-2) enhancer element (see Tuan, An erythroidspecific, development stage-independent enhancer far upstream of thehuman “β-like globin” genes, Proc. Natl. Acad. Sci. 86 (1989) 2554-2559)from the locus control region (LCR) from the human β-globin gene cluster(vHS2-^(-A)γ-globin) were directly injected into C57B1/6 mice.

[0050] Approximately 1×10¹⁰ particles of the vHS2βp-^(A)γ-globin r-viruswere injected i.v. as described in Example 1. Seven weeks p.i., thevarious organs were obtained and analyzed for the presence of ther-viral genome using the human β-globin promoter(5′GATGGTATGGGGCCAAGAGA)-and ^(A)γ-globin gene(5′-GGGTTTCTCCTCCAGCATCT)-specific oligodeoxynucleotide primer pair.Liver tissues obtained from a mock-injected mouse was also included as anegative control. The Southern blot results are shown in FIG. 2. Thearrow indicates the 354-bp human γ-globin-specific DNA fragment.

[0051] We then investigated copy number of the vHS2-βp-^(A)γ-globinvector in liver cells. Equivalent amounts of DNA isolated from the liverof mock-injected and vHS2-βp-^(A)γ-globin virus-injected mice were usedin a semi-quantitative PCR amplification assay using either the humanβ-globin promoter-^(A)γ-globin gene-specific oligodeoxynucleotideprimers or the mouse β-actin-specific oligodeoxynucleotide primers.Approximately equivalent amounts of liver tissue from each animals werelysed in a buffer containing 10 mM Tris.HCl/50 mM KCl/2.5 mM MgCl₂/0.5%Tween-20/100 μg proteinase K per ml at 55° C. overnight. The lysateswere heated at 90° C. for 10 min to inactivate proteinase K, and 5 μl ofeach sample was subjected to a 30-cycle PCR amplification with the twosets of primer-pairs under identical conditions. The primers foramplifying the transduced human globin gene sequences were the same asthose described in Example 1 and the primer sequences for the mouseβ-actin gene were as follows: 5′-ACCTTCAACACCCCAGCCAT and5′-TCAGGCAGCTCATAGCTCTT. The primers were designed to yield a 354-bp DNAfragment from each sequence. The PCR reactions were performed in thepresence of 2 μCi [α-³²P]dCTP (sp. act. 800 Ci/mmol) in each reactionmix. Ten per cent of the DNA products from the human globin gene and a15-fold diluted samples from the β-actin gene amplification reactionswere analyzed on 6% polyacrylamide gels and autoradiographed. Therelative intensities of the corresponding bands were determined byscanning the autoradiograms using the Photoshop 3.0 program. Thetransduced globin gene was detected in approximately 4% of liver cellsseven weeks p.i. See FIG. 3.

EXAMPLE 3

[0052] We next examined whether the lacZ gene delivered by directinjection of the r-AAV was transcriptionally active. Livers frommock-injected and vCMVp-lacZ-injected C57B1/6 mice were obtained oneweek p.i., and cryopreserved. Tissue sections were fixed and stainedwith 5Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (XGal) as describedin Cheng, Separable Regulatory Elements Governing Myogenin Transcriptionin Mouse Embryogenesis, Science 261 (1993) 215-218 and visualized undera light microscope.

[0053] Livers were obtained one week p.i. and frozen immediately iniso-pentane at −40° C. Sections of 15 μm were prepared using a cryostatand fixed in a solution containing 2% formaldehyde/0.2%para-formaldehyde in phosphate-buffered saline (PBS, 135 mM NaCl/2.5 mMKCl/8 mM Na₂HPO₄/0.6 mM KH₂PO₄/0.55 mM dextrose/liter) for 5 min on ice,washed twice with PBS and stained overnight at 37° C. in a solutioncontaining 5 mM K₃Fe(CN)₆/5 mM K₄Fe(CN)₆/1 mM MgCl₂/1 mg XGal in 1 ml ofPBS, as described previously in Cheng, Separable regulatory elementsgoverning myogenin transcription in mouse embryogenesis. Science 261(1993) 215-218. Tissue sections were visualized under a light microscope(magnification×40). No expression of the transgene occurred in livercells from mock-injected animal. Expression of the lacZ gene was readilydetected in liver hepatocytes.

EXAMPLE 4

[0054] Co-transfection of an rAAV vector containing the AAV ITRs and thenucleic acid sequence encoding a therapeutic molecule and a helperplasmid containing the necessary rep and cap functions into adenovirus-2(Ad2) infected 293 cells was expected to eliminate homologousrecombination events leading to the production of contaminatingwild-type (wt) AAV during the production of recombinant vector stocks.However, contaminating “wild type-like AAV” particles have been observedin such stocks ranging from 0.1% to 10%.

[0055] To determine the mechanism of generation of contaminating wt AAV,stocks were amplified through four successive round of co-infection withAd2 in 293 cells. Low molecular weight DNA fragments were isolated,digested with BalI restriction endonuclease and molecularly cloned intoa pBlueScript plasmid vector. AAV sequence-positive clones weresubjected to nucleotide sequencing using T3 and T7 primers. Nucleotidesequence analysis of 12 independent clones revealed that most of therecombination events leading to the contaminating wt AAV involved 10nucleotides in the AAV D-sequence distal to viral hairpin structures. Inaddition, by analyzing 22 different clones generated with a helperplasmid that lacks the Ad2 ITRs, we observed only a limited number ofrecombination sites and concluded that Ad2 ITRs play a role inillegitimate recombination with the AAV-ITRs that leads to generation ofbiologically active wild type-like AAV. Consequently, by removing theAd2 ITRs from the helper plasmid, nearly 5-fold reduction in theillegitimate recombination frequency can be achieved.

[0056] The first 10 nucleotides in the D-sequence proximal to the AAVhairpin structures are essential for successful replication andencapsidation of the viral genome. See, Wang, J Virol 71:3077-82 (1997).In each of the recombinant junctions sequenced, the same 10 nucleotideswere retained. By deleting the distal 10 nucleotides in the D-sequencein the next generation of AAV vectors, the generation of the wt AAV-likeparticles in recombinant AAV vectors stocks can be redueced oreliminated. See Example 5 below for production of such vectors.

EXAMPLE 5

[0057] Four recombinant AAV vectors, pD-5, pD-10, pD-15 and pD-20, wereconstructed as follows. Plasmid pXS-22 can be employed as startingmaterial. The plasmid pXS-22 can be obtained from a public depository orconstructed following the methods described in Wang, J. Mol. Biol. 250(1995) 573-580 using pSub201 as starting material. Plasmid pXS-22contains only the right ITR (inverted terminal repeat): one hairpin andone D sequence. The D-sequence is that part of the AAV ITR which is notinvolved in HP formation. See Wang, supra. The D-sequence can bereplaced by a substitute (S) sequence as described in Wang, J Virol. 70(1996) 1668-1677. The nucleotide sequences are as follows:

[0058] D-sequence

[0059] 5′ CTCCA TCACT AGGGG TTCCT

[0060] 3′ GAGGT AGTGA TCCCC AAGGA 5′

[0061] S-sequence

[0062] 5′ CCAA TATTA GATCT GATAT CA 3′

[0063] 3′ GGTT ATAAT CTAGA CTATA GTGAT C 5′

[0064] Four additional oligonucleotide sequences were synthesized whichcontained selected nucleotides identical to the authentic or nativeD-sequence in place of nucleotides in the S-sequence. These fouroligonucleotides are:

[0065] D-5 oligonucleotide

[0066] 5′ CCAA CTCCA GATCT GATAT CACTT 3′

[0067] 3′ GGTT GAGGT CTAGA CTATA GTGAA GATC

[0068] D-10 oligonucleotide

[0069] 5′ CCAA CTCCA TCACT GATAT CACTT 3′

[0070] 3′ GGTT GAGGT AGTGA CTATA GTGAA GATC 5′

[0071] D-15 oligonucleotide

[0072] 5′ CCAA CTCCA TCACT AGGGG CACTT 3′

[0073] 3′ GGTT GAGGT AGTGA TCCCC GTGAA GATC 5′

[0074] D-20 oligonucleotide

[0075] 5′ CCAA CTCCA TCACT AGGGG TTCCT 3′

[0076] 3′ GGTT GAGGT AGTGA TCCCC AAGGA GATC 5′

[0077] The selected nucleotides conforming to the authentic, native,D-sequence in the AAV ITR are indicated above in bold.

[0078] The D-5, D-10, D-15 and D-20 oligonucleotide sequences were eachinserted between the Xba I and Bal I sites of plasmid pXS-22, which isdescribed in Wang, J. Mol. Biol. 250 (1995) 573-580 and J Virol. 70(1996) 1668-1677. The resulting four plasmids were named pXS-64D-5,pXS-64D-10, pXS-64D-15 and pXS-64D-20 respectively. The bluntedClaI-PvuII fragments from pXS-64D-5, pXS-64D-10, pXS-64D-15 andpXS-64D-20 were then excised and ligated between the ClaI and XbaI sitesof these plasmids to generate plasmids pD-5, pD-10, pD-15 and pD-20respectively containing the D-5, D-10, D-15 and D-20 oligonucleotidesequences in place of the S sequences in both ITRs.

[0079] Each of the four foregoing recombinant AAV vectors, pD-5, pD- 10,pD-15 and pD-20 may be employed in the methods of the invention. We havedetermined that to optimize packaging 10 of the native D-nucleotides aresufficient. The most preferred native 10 D-nucleotides are thoseincluded in the pD-10 vector and indicated in bold in the D-10oligonucleotide sequence above. The pD-15 and pD-20 vectors, or theirrespective indicated oligonucleotides (see above), may be used but theycontain extra, unnecessary nucleotides that would advantageously beeliminated in order to allow for more space in the AAV vector fornucleotides encoding the desired therapeutic molecule. The pD-5 vectorworks, but with less efficiency. Consequently, the absolute minimalnecessary sequence is the 5 nucleotide sequence enumerated in bold inthe D-5 oligonucleotide sequence above and contained in the pD-5 vector.The pD-10 vector allows for the insertion of an additional 106nucleotides.

[0080] Nucleic acid sequences encoding therapeutic molecules can beligated between the ITRs of these vectors using known techniques. Thevectors or virions may be formulated into pharmaceutical compositionsfor administration in human or other mammalian patients.

[0081] Plasmid pXS-22 was deposited on Sep. 10, 1996 with the ATCC,12301 Parklawn Drive, Rockville, Md., USA under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for Purposes of Patent Procedure. The Accession Number is97710. This deposit assures maintenance of a viable culture for 30 yearsfrom the date of deposit. The organism(s) deposited will be madeavailable by the ATCC under the terms of the Budapest Treaty, andsubject to an agreement between applicant and the ATCC that assuresunrestricted availability upon issuance of the pertinent U.S. patent.This deposit is provided as convenience to those of skill in the art,and is not an admission that a deposit is required under 35 U.S.C. 112.The nucleic acid sequence of this deposit, as well as the amino acidsequence of the polypeptide(s) encoded thereby, are incorporated hereinby reference and should be referred to in the event of an error in thesequence described herein. A license may be required to make, use, orsell the deposited materials, and no such license is granted hereby.

[0082] All patents, patent publications, patent applications andscientific articles mentioned in this specification are hereinincorporated by reference. The invention now being fully described, itwill be apparent to one of ordinary skill in the art that many changesand modifications can be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A method for realizing liver specific delivery ofa therapeutic molecule in a mammalian patient comprising administeringto said mammalian patient a therapeutically effective amount of an AAVvector containing said therapeutic molecule.
 2. A method according toclaim 1 wherein said therapeutic molecule is a nucleic acid sequenceencoding a secretory protein, an antisense molecule or a ribozyme.
 3. Amethod according to claim 1 wherein the AAV vector is selected from thegroup consisting of pD-5, pD-10, pD-15, pD-20.
 4. A method according toclaim 1 wherein said AAV vector is administered by intravenous orintraportal injection.
 5. A method according to claim 1 wherein said AAVvector is administered ex vivo.
 6. A method according to claim 1 whereinsaid AAV vector is administered by direct injection.
 7. A methodaccording to claim 1 wherein said therapeutic molecule is a nucleic acidencoding the LDL receptor.
 8. A method according to claim 1 wherein saidAAV vector additionally contains a liver specific promoter.
 9. A methodaccording to claim 8 wherein said AAV vector additionally contains aliver specific enhancer.
 10. A method according claim 8 wherein saidpromoter is selected from the group consisting of the hepatitis B virusX gene promoter, the hepatitis B virus core protein promoter, the AFPgene promoter, the albumin gene promoter, the α-1 antitrypsin genepromoter, the fibrinogen gene promoter, the APO-A1 gene promoter and thepromoter genes for liver transference enzymes.
 11. A method according toclaim 10 wherein said AAV vector additionally contains a liver specificenhancer.
 12. A hybrid helper AAV vector comprising a DNA sequenceencoding hepatitis B virus surface antigen or a functional fragmentthereof linked to a DNA sequence encoding the AAV VP-1 protein to form achimeric DNA sequence.
 13. A recombinant AAV vector comprising two AAVITRs (inverted terminal repeats) in which the native D-sequences of eachof said ITRs are modified by the substitution of nucleotides such thatat least 5 native nucleotides and up to 18 native nucleotides areretained and the remaining nucleotides of the D-sequence are deleted orreplaced with non-native nucleotides.
 14. A recombinant AAV vectoraccording to claim 13 wherein said at least 5 native nucleotides are 5′CTCCA 3′.
 15. A recombinant AAV vector comprising two AAV ITRs (invertedterminal repeats) in which the native D-sequences of each of said ITRsare modified by the substitution of nucleotides such that at least 10native nucleotides up to 18 native nucleotides are retained and theremaining nucleotides of the D-sequence are deleted or replaced withnon-native nucleotides.
 16. A recombinant AAV vector comprising two AAVITRs (inverted terminal repeats) in which the native D-sequences of eachof said ITRs are modified by the substitution of nucleotides such that10 native nucleotides are retained and the remaining nucleotides of theD-sequence are deleted or replaced with non-native nucleotides.
 17. Arecombinant AAV vector according to claim 16 wherein said 10 nativenucleotides comprise nucleotides 5′ CTCCA 3′ and five other nativenucleotides of said D-sequence.
 18. A recombinant AAV vector selectedfrom the group consisting of pD-5, pD-10, pD-15 and pD-20.